VEGF binds to a receptor present in vascular endothelial cells to promote growth of the endothelial cells, thereby promoting angiogenesis. VEGF is therefore attracting attention from the viewpoint of its association with diseases in which angiogenesis plays an important role. For example, VEGF is reported to show an elevated serum level in patients with solid tumors. Thus, substances that bind to VEGF are expected to be sensor elements useful for diagnosis of various diseases accompanied by angiogenesis.
Measurement of a test substance such as a protein in a sample is carried out mainly by an immunoassay at present. Various immunoassay methods are known and practically used, and any of these methods uses an antibody specific to the test substance. Although antibodies specific to test substances can be prepared by conventional methods, the methods are laborious, and specific antibodies are therefore expensive.
On the other hand, aptamers, which are polynucleotide molecules that specifically bind to arbitrary molecules, are known. Since total chemical synthesis of aptamers can be carried out using a commercially available nucleic acid synthesizer, aptamers are much less expensive than specific antibodies, and can be easily modified. Therefore, their practical use as sensor elements has been expected. An aptamer that specifically binds to a desired target molecule can be prepared by a method called SELEX (Systematic Evolution of Ligands by EXponential Enrichment) (Non-patent Document 1). In this method, the target molecule is immobilized on a carrier, and a nucleic acid library composed of nucleic acid having vast kinds of random base sequences is added thereto, followed by recovering nucleic acid binding to the target molecule. The recovered nucleic acid is amplified by PCR, and then added again to the carrier on which the target molecule is immobilized. By repeating this process about 10 times, aptamers having high binding capacities to the target molecule are concentrated, and their sequences are determined to obtain aptamers that recognize the target molecule. The nucleic acid library can be easily prepared by randomly binding nucleotides using an automated chemical synthesizer for nucleic acid. Thus, by a method which positively utilizes contingency by using a nucleic acid library containing random base sequences, an aptamer which specifically binds to an arbitrary target substance can be prepared.
The present inventors previously succeeded in obtaining an aptamer that binds to VEGF by SELEX which utilizes a method comprising: binding a desired target substance to nucleic acid in a nucleic library in the presence of a carrier on which a non-target substance is immobilized; recovering only nucleic acid bound to the desired target substance; amplifying the recovered nucleic acid by PCR; obtaining single-stranded nucleic acid from the amplified product to provide a nucleic acid library; bringing the nucleic acid library into contact with an area on which the target substance is immobilized; recovering nucleic acid bound to the solid phase; similarly amplifying the recovered nucleic acid by PCR; obtaining single-stranded nucleic acid therefrom to provide a nucleic acid library; bringing the nucleic acid library again into contact with areas on each of which the target substance or the non-target substance is immobilized; and then repeating this cycle in the same manner (Patent Document 1).